Skip to main content
SpringerLink
Log in

Modern Techniques for DNA and RNA Assessments

  • Chapter
  • First Online:
Success in Academic Surgery: Basic Science

Part of the book series: Success in Academic Surgery ((SIAS))

  • 1234 Accesses

Abstract

The role of molecular genetics and genomics has risen to great importance in modern medicine. Medicine has seen an exponential growth in methods used to diagnose genetic aberrations leading to better understanding of disease processes. Examples of the advancement have impacted all medical and surgical specialties including improved prenatal screening for rare diseases, pharmacogenetics, and genetic profiling of malignancies. Improved understanding of disease processes at the genetic level has led to improvements in diagnosis, prognosis, and in some cases treatment. Advancements in laboratory methods for RNA and DNA analysis and sequencing have made possible our current understanding of both simple and complex pathology at its fundamental level. Surgeons, physicians, and healthcare professionals must develop an understanding of the methods of RNA and DNA analysis. Reviewed here are the basics and advancements in genetic and genomic analysis that are used in research and clinical medicine.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 69.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature. 2004;431(7011):931–45.

    Article  Google Scholar 

  2. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature. 2001;409(6822):860–921.

    Article  PubMed  CAS  Google Scholar 

  3. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, et al. The sequence of the human genome. Science. 2001;291(5507):1304–51.

    Article  PubMed  CAS  Google Scholar 

  4. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975;98(3):503–17.

    Article  PubMed  CAS  Google Scholar 

  5. Mathews CK, van Holde KE, Appling DR, Anthony-Cahill SJ. Biochemistry. 4th ed. Toronto: Prentice Hall; 2012.

    Google Scholar 

  6. Maxam AM, Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977;74(2):560–4.

    Article  PubMed  CAS  Google Scholar 

  7. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977;74(12):5463–7.

    Article  PubMed  CAS  Google Scholar 

  8. Smith LM, Sanders JZ, Kaiser RJ, Hughes P, Dodd C, Connell CR, et al. Fluorescence detection in automated DNA sequence analysis. Nature. 1986;321(6071):674–9.

    Article  PubMed  CAS  Google Scholar 

  9. Prober JM, Trainor GL, Dam RJ, Hobbs FW, Robertson CW, Zagursky RJ, et al. A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science. 1987;238(4825):336–41.

    Article  PubMed  CAS  Google Scholar 

  10. Cohen AS, Najarian DR, Paulus A, Guttman A, Smith JA, Karger BL. Rapid separation and purification of oligonucleotides by high-performance capillary gel electrophoresis. Proc Natl Acad Sci U S A. 1988;85(24):9660–3.

    Article  PubMed  CAS  Google Scholar 

  11. Luckey JA, Drossman H, Kostichka AJ, Mead DA, D’Cunha J, Norris TB, et al. High speed DNA sequencing by capillary electrophoresis. Nucleic Acids Res. 1990;18(15):4417–21.

    Article  PubMed  CAS  Google Scholar 

  12. Stranneheim H, Lundeberg J. Stepping stones in DNA sequencing. Biotechnol J. 2012;7(9):1063–73.

    Article  PubMed  CAS  Google Scholar 

  13. Melamede RJ. Automatable process for sequencing nucleotide. US Patent 4863849. 1985.

    Google Scholar 

  14. Nyren P. The history of pyrosequencing. Methods Mol Biol. 2007;373:1–14.

    Article  PubMed  CAS  Google Scholar 

  15. Ronaghi M, Uhlen M, Nyren P. A sequencing method based on real-time pyrophosphate. Science. 1998;281(5375):363, 365.

    Article  PubMed  CAS  Google Scholar 

  16. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature. 2005;437(7057):376–80.

    PubMed  CAS  Google Scholar 

  17. Rothberg JM, Leamon JH. The development and impact of 454 sequencing. Nat Biotechnol. 2008;26(10):1117–24.

    Article  PubMed  CAS  Google Scholar 

  18. Bennett ST, Barnes C, Cox A, Davies L, Brown C. Toward the $1000 human genome. Pharmacogenomics. 2005;6(4):373–82.

    Article  PubMed  CAS  Google Scholar 

  19. Balasubramanian S, Bentley D. Polynucleotide arrays and their use in sequencing. Patent WO 01/157248. 2001.

    Google Scholar 

  20. Shendure J, Ji H. Next-generation DNA sequencing. Nat Biotechnol. 2008;26(10):1135–45.

    Article  PubMed  CAS  Google Scholar 

  21. Bentley DR. Whole-genome re-sequencing. Curr Opin Genet Dev. 2006;16(6):545–52. Epub 2006 Oct 18.

    Article  PubMed  CAS  Google Scholar 

  22. Li R, Fan W, Tian G, Zhu H, He L, Cai J, et al. The sequence and de novo assembly of the giant panda genome. Nature. 2010;463(7279):311–7. Epub Dec 13 2009.

    Article  PubMed  CAS  Google Scholar 

  23. Li R, Zhu H, Ruan J, Qian W, Fang X, Shi Z, et al. De novo assembly of human genomes with massively parallel short read sequencing. Genome Res. 2010;20(2):265–72.

    Article  PubMed  CAS  Google Scholar 

  24. Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, Rosenbaum AM, et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science. 2005;309(5741):1728–32.

    Article  PubMed  CAS  Google Scholar 

  25. Metzker ML. Sequencing technologies-the next generation. Nat Rev Genet. 2010;11:31–46.

    Article  PubMed  CAS  Google Scholar 

  26. Pourmand N, Karhanek M, Persson HHJ, Webb CD, Lee TH, Zahradnlkova A, et al. Direct electrical detection of DNA synthesis. Proc Natl Acad Sci U S A. 2006;103(17):6466–70.

    Article  PubMed  CAS  Google Scholar 

  27. Rothberg JM, Hinz W, Rearick TM, Schultz J, Mileski W, Davey M, et al. An integrated semiconductor device enabling non-optical genome sequencing. Nature. 2011;475(7356):348–52.

    Article  PubMed  CAS  Google Scholar 

  28. Toumazou C, Premanode B, Shepherd L. Signal processing circuit comprising ion sensitive field effect transistor and method of monitoring a property of a fluid. DNA Electronics. US Patent 20080265985. 2008.

    Google Scholar 

  29. Mellmann A, Harmsen D, Cummings CA, Zentz EB, Leopold SR, Rico A, et al. Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology. PLoS One. 2011;6(7):e22751.

    Article  PubMed  CAS  Google Scholar 

  30. Rohde H, Qin J, Cui Y, Li D, Loman NJ, Hentschke M, et al. Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4. N Engl J Med. 2011;365(8):718–24. Epub Jul 27 2011.

    Article  PubMed  CAS  Google Scholar 

  31. Hellman LM, Fried MG. Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protoc. 2007;2(8):1849–61.

    Article  PubMed  CAS  Google Scholar 

  32. Lane D, Prentki P, Chandler M. Use of gel retardation to analyze protein-nucleic acid interactions. Microbiol Rev. 1992;56(4):509–28.

    PubMed  CAS  Google Scholar 

  33. Cai Y, Huang H. Advances in the study of protein-DNA interaction. Amino Acids. 2012;43:1141–6.

    Article  PubMed  CAS  Google Scholar 

  34. Brenowitz M, Senear DF, Shea MA, Ackers GK. Quantitative DNase footprint titration: a method for studying protein-DNA interactions. Methods Enzymol. 1986;130:132–81.

    Article  PubMed  CAS  Google Scholar 

  35. Orlando V. Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends Biochem Sci. 2000;25(3):99–104.

    Article  PubMed  CAS  Google Scholar 

  36. Horak CE, Snyder M. ChIP-chip: a genomic approach for identifying transcription factor binding sites. Methods Enzymol. 2002;350:469–83.

    Article  PubMed  CAS  Google Scholar 

  37. Robertson G, Hirst M, Bainbridge M, Bilenky M, Zhao Y, Zeng T, et al. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods. 2007;4(8):651–7.

    Article  PubMed  CAS  Google Scholar 

  38. Brown D, Brown J, Kang C, Gold L, Allen P. Single-stranded RNA recognition by the bacteriophage T4 translational repressor, regA. J Biol Chem. 1997;272(23):14969–74.

    Article  PubMed  CAS  Google Scholar 

  39. Jahanmir J, Haggar BG, Hayes JB. The scanning probe microscope. Scanning Microsc. 1992;6(3):625–60.

    PubMed  CAS  Google Scholar 

  40. Binnig G, Quate CF, Gerber C. Atomic force microscope. Phys Rev Lett. 1986;56(9):930–3.

    Article  PubMed  Google Scholar 

  41. Binnig G, Rohrer H, Gerber C, Weibel E. Tunneling through a controllable vacuum gap. Appl Phys Lett. 1982;40:178–80.

    Article  CAS  Google Scholar 

  42. Betzig E, Trautman JK, Harris TD, Weiner JS, Kostelak RL. Breaking the diffraction barrier: optical microscopy on a nanometric scale. Science. 1991;251(5000):1468–70.

    Article  PubMed  CAS  Google Scholar 

  43. Hoa XD, Kirk AG, Tabrizian M. Towards integrated and sensitive surface plasmon resonance biosensors: a review of recent progress. Biosens Bioelectron. 2007;23(2):151–60.

    Article  PubMed  CAS  Google Scholar 

  44. Dolezel J, Vrana J, Safar J, Bartos J, Kubalakova M, Simkova H. Chromosomes in the flow to simplify genome analysis. Funct Integr Genomics. 2012;12:397–416.

    Article  PubMed  CAS  Google Scholar 

  45. Brown TA. Genomes 3. 3rd ed. New York: Garland Science; 2007.

    Google Scholar 

  46. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterotrophic gene lin-4 encodes small RNAs with antisense complimentary to lin-14. Cell. 1993;75(5):843–54.

    Article  PubMed  CAS  Google Scholar 

  47. Girard A, Sachidanandam R, Hannon GJ, Carmell MA. A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature. 2006;442(7099):199–202.

    PubMed  Google Scholar 

  48. Aravin A, Gaidatzis D, Pfeffer S, Lagos-Quintana M, Landgraf P, Iovino N, et al. A novel class of small RNAs bind to MILI protein in mouse testes. Nature. 2006;442(7099):203–7.

    PubMed  CAS  Google Scholar 

  49. Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, et al. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci U S A. 2009;106(28):11667–72.

    Article  PubMed  CAS  Google Scholar 

  50. Alwine JC, Kemp DJ, Stark GR. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977;74(12):5350–4.

    Article  PubMed  CAS  Google Scholar 

  51. Dvorák Z, Pascussi JM, Modrianský M. Approaches to messenger RNA detection – comparison of methods. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2003;147(2):131–5.

    Article  PubMed  Google Scholar 

  52. Gall JG, Pardue ML. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci U S A. 1969;63(2):378–83.

    Article  PubMed  CAS  Google Scholar 

  53. Lin L, Lloyd RV. In situ hybridization: methods and applications. J Clin Lab Anal. 1997;11:2.

    Article  Google Scholar 

  54. Schena M, Shalon D, Davis RW, Brown PO. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science. 1995;270(5235):467–70.

    Article  PubMed  CAS  Google Scholar 

  55. Churchill GA. Fundamentals of experimental design for cDNA microarrays. Nat Genet. 2002;32(Suppl):490–5.

    Article  PubMed  CAS  Google Scholar 

  56. Pease AC, Solas D, Sullivan EJ, Cronin MT, Holmes CP, Fodor SP. Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc Natl Acad Sci U S A. 1994;91(11):5022–6.

    Article  PubMed  CAS  Google Scholar 

  57. Nuwaysir EF, Huang W, Albert TJ, Singh J, Nuwaysir K, Pitas A, et al. Gene expression analysis using oligonucleotide arrays produced by maskless photolithography. Genome Res. 2002;12(11):1749–55.

    Article  PubMed  CAS  Google Scholar 

  58. Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2006;1(3):1559–82.

    Article  PubMed  CAS  Google Scholar 

  59. VanGuilder HD, Vrana KE, Freeman WM. Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques. 2008;44(5):619–26.

    Article  PubMed  CAS  Google Scholar 

  60. Wilhelm BT, Landry JR. RNA-seq-quantitative measurement of expression through massive parallel RNA-sequencing. Methods. 2009;48:249.

    Article  PubMed  CAS  Google Scholar 

  61. Quackenbush J. Microarray data normalization and transformation. Nat Genet. 2002;32(Suppl):496–501.

    Article  PubMed  CAS  Google Scholar 

  62. Faith JJ, Hayete B, Thaden JT, Mongo I, Wierzbowski J, Cottarel G, et al. Large-scale mapping and validation of Escherichia coli transcriptional regulation from a compendium of expression profiles. PLoS Biol. 2007;5(1):e8.

    Article  PubMed  Google Scholar 

  63. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc. 1995;57:289.

    Google Scholar 

  64. Storey JD, Tibshirani R. Statistical significance for genome-wide studies. Proc Natl Acad Sci U S A. 2003;100(16):9440–5.

    Article  PubMed  CAS  Google Scholar 

  65. Barrett T, Troup DB, Wilhite SE, Ledoux P, Rudnev D, Evangelista C, et al. NCBI GEO: archive for high-throughput functional genomic data. Nucleic Acids Res. 2009;37(Database issue):D885–90.

    Google Scholar 

  66. Dai M, Wang P, Boyd AD, Kostov G, Athey B, Jones EG, et al. Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data. Nucleic Acids Res. 2005;33(20):e175.

    Article  PubMed  Google Scholar 

  67. Subramanian A, Tamayo P, Mootha VK, Mookherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545–50. Epub 2005 Sep 30.

    Article  PubMed  CAS  Google Scholar 

  68. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet. 2003;34(3):267–73.

    Article  PubMed  CAS  Google Scholar 

  69. Lamb J, Crawford ED, Peck D, Modell JW, Blat IC, Wrobel MJ, et al. The connectivity map: using gene-expression signatures to connect small molecules, genes, and disease. Science. 2006;313(5795):1929–35.

    Article  PubMed  CAS  Google Scholar 

  70. Dennis Jr G, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, et al. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 2003;4(5):P3. Epub 2003 Apr 3.

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of General Surgery, University of Pittsburgh Medical Center, F1200 PUH, 200 Lothrop Street, Pittsburgh, PA, 15213, USA

    Benjamin Kautza MD & Brian S. Zuckerbraun MD

Authors
  1. Benjamin Kautza MD
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Brian S. Zuckerbraun MD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Brian S. Zuckerbraun MD .

Editor information

Editors and Affiliations

  1. Professor of Surgery, Vice Chair of Research, Edward G. Elcock Professor of Surgical Research, Department of Surgery, Northwestern University, Chicago, IL, USA, and Co-Chief, Peripheral Vascular Surgery, Jesse Brown VA Medical Center, Chicago, Illinois, USA

    Melina R. Kibbe

  2. Division of Cardiothoracic Surgery, Baylor College of Medicine, and Department of Cardiovascular Surgery, Texas Heart Institute, St. Luke's Episcopal Hospital, Houston, Texas, USA

    Scott A. LeMaire

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer-Verlag London

About this chapter

Cite this chapter

Kautza, B., Zuckerbraun, B.S. (2014). Modern Techniques for DNA and RNA Assessments. In: Kibbe, M., LeMaire, S. (eds) Success in Academic Surgery: Basic Science. Success in Academic Surgery. Springer, London. https://doi.org/10.1007/978-1-4471-4736-7_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-4471-4736-7_8

  • Published:

  • Publisher Name: Springer, London

  • Print ISBN: 978-1-4471-4735-0

  • Online ISBN: 978-1-4471-4736-7

  • eBook Packages: MedicineMedicine (R0)

Publish with us

Policies and ethics

Search

Navigation